1. Field of the Invention
All publications or patents mentioned in this specification are herein incorporated by reference.
This invention relates to a unique nucleic acid hybridization probe and method for the rapid detection of typhoid fever bacteria.
2. Prior Disclosure
Diarrheal diseases caused by enteric bacteria are still a major cause of illness and death worldwide, especially among infants and young children in developing nations. Also, these maladies are an important military problem in deployed soldiers. Although the incidence of diarrheal disease is highest in tropical countries, geography is not as important a factor as socioeconomic conditions; e.g. as manifested by drinking water purity, sewage disposal methods, and the availability of balanced diets. Some enteric diseases are short-lived, self-limiting and result in a mild gastroenteritis (e.g. certain Salmonella serotypes). In contrast, typhoid fever, caused by Salmonella typhi, is a prolonged, generalized, and usually serious infection of humans of all age groups. Similar enteric diseases are caused by related bacteria such as Salmonella paratyphi A, B, and C and by other Salmonella serotypes.
All strains of Salmonella typhi and S. paratyphi C, as well as a few atypical but genetically related Citrobacter and Salmonella strains, are capable of synthesizing a capsular antigen termed Vi for virulence (Edwards, P. R., and W. H. Ewing, 1972, Identification of Enterobacteriaceae, 3rd Edition pages 146-207, Burgess Publishing Company, Minneapolis). This galactosamine uronic acid polymer (i.e. the Vi antigen) has been associated with the virulence of S. typhi (Felix, A., S. S. Bhatnagar, and R. M. Pitt, 1934, Observations on the Properties of the Vi Antigen of B. typhosus, Br. J. Exp. Pathol. 15:346-354; and Heyns, K., G. Kiessling, W. Lindenberg, H. Paulsen, and M. E. Webster, 1959, d-Galaktosaminuronsaure (2-Amino-2-desoxy-D-galakturonsaure) als Baustein des Vi-Antigens, Chem. Ber. 92:2435-2437). Two separate chromosomal loci necessary for Vi antigen expression, viaA and viaB, have been identified in genetic studies of S. typhi (Johnson, E. M., B. Krauskopf, and L. S. Baron, 1965, Genetic mapping of Vi and somatic antigenic determinants in Salmonella, J. Bacteriol. 90:302- 308; and Johnson, E. M. B., B. Krauskopf, and L. S. Baron, 1966, Genetic analysis of the viaA-his chromosomal region in Salmonella, J. Bacteriol. 92:1457-1463). The viaB region appears to encode the structural genes for this antigen (Johnson, E. M., B. Krauskopf, and L. S. Baron, 1965, Genetic mapping of Vi and somatic antigenic determinants in Salmonella, J. Bacteriol. 90:302-308). Analogous and presumably allelic chromosomal sites have been identified in S. paratyphi C (Snellings, N. J., E. M. Johnson, and L. S. Baron, 1977, Genetic basis of Vi antigen expression in Salmonella paratyphi C. J. Bacteriol. 131:57-62) and in some strains of Citrobacter freundii (Snellings, N. J., E. M. Johnson, D. J. Kopecko, H. H. Collins, and L. S. Baron, 1981, Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii, J. Bacteriol. 145:1010-1017). Although the expression of the Vi antigen is relatively stable in S. typhi, Vi-positive Citrobacter strains exhibit a rapid, reversible transition between forms that express the Vi antigen and forms that appear not to express it, referred to as non-Vi or W forms (Baron, L. S., D. J. Kopecko, S. M. McCowen, N. J. Snellings, E. M. Johnson, W. C. Reid, and C. A. Life, 1982, Genetic and molecular studies on the regulation of a typical citrate utilization and variable Vi antigen expression in enteric bacteria, pages 175-194, In Hollaender (Editor), Genetic Engineering of Microorganisms For Chemicals, Plenum Press, NY; and Snellings, N. J., E. M. Johnson, D. J. Kopecko, H. H. Collins, and L. S. Baron, 1981, Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii, J. Bacteriol. 3 145 1010-1017).
Proper chemotherapeutic treatment of typhoid or related enteric fever disease in many cases is only instituted following the proper identification of the causative agent. The standard biochemical and serological identification of enteric bacteria from fecal or blood specimens generally requires 24 to 48 hours even with the most up-to-date clinical microbiology facilities. The absence of these facilities in areas of military troop deployment and in underdeveloped countries prevents the proper epidemiological identification of diseases and the administration of appropriate chemotherapeutic regimens A rapid method, which could be utilized in remote, ill-equipped areas, for the identification of specific enteric bacteria would be of obvious benefit to mankind. Several scientific groups have developed deoxyribonucleic acid (i.e. DNA) hybridization techniques, disclosed in U.S. Pat. Nos. 4,358,535 (Falkow, et al.) and 4,139,346 (Rabbani), and specific immunological procedures for the rapid identification of bacteria viruses and other organisms in culture specimens. Thus, there are several basic concepts available around which one can design a rapid diagnostic detection tool. Notwithstanding these readily available data, it takes considerable ingenuity to develop a bacterial identification assay that is differentially specific, rapid and inexpensive, and which can he conducted in remote areas with little equipment. For these reasons, relatively few rapid diagnostic assays are broadly applicable.